The initiation of X inactivation during early embryogenesis and the subsequent maintenance of the inactive X condition throughout development are central issues in the X-inactivation process. These features and the succeeding reactivation during oogenesis are unique developmental changes which produce the coordinate regulation of an entire X chromosome. This proposal is a broadly based approach to studying the X-inactivation process in the mouse. Our experimental strategies draw upon using X-chromosome gene variation we have recovered from a wide sampling of the mouse gene pool to follow the expression of specific X chromosomes. We have extended this strategy to identifying DNA variation which can be used to follow DNA methylation on active and inactive X-chromosome genes. We are now in a position to extend our use of naturally occurring variation of X chromosome genes to efficiently screen mutagen treated stocks for additional X chromosome markers and to identify mutations which provide an opportunity to pursue a genetic analysis of X-chromosome regulation during the inactivation process. We are also extending the use of biochemical markers of X-chromosome genes to examine the expression of X-chromosome genes in X;autosome translocations. The expression of genes which are either proximal or distal to the translocation will provide some insight into the site(s) which establish X-chromosome identity in the inactivation process. This proposal also describes experiments to study X-chromosome reactivation in embryonal carcinoma cell-somatic cell hybrids. This work will provide important information about the inactivation and reactivation process and it offers an opportunity to develop new experimental approaches to studying X-chromosome regulation.